1. Technical Field
The present invention relates to a carrier for binding of antiphospholipid antibodies, an immunoassay method using the same, a kit therefor, and a fraction and a protein obtained from serum or plasma which can be used in the method for immunoassay. In particular, the present invention relates to determination of autoantibodies specifically appeared in patients with antiphospholipid syndrome.
2. Background Art
A living body causes immune response against exogenous foreign matters such as pathogenic viruses, bacteria, fungi, parasites, etc. invaded into a living body, which functions to expel the foreign matters. The phenomenon is generally classified into the following two groups: humoral immunity in which antibodies participate and cellular immunity in which immunocompetent cells directly participate, thereby to expel the foreign matters. In diseases collectively referred to as autoimmune diseases, however, recognition mechanism of self or non-self does not work correctly but humoral or cellular immune response occurs against their own cells or tissues. As a representative clinical case in which antibodies reactive with self components (autoantibodies) appear, there is systemic lupus erythematosus (SLE). It is noted that anti-single stranded DNA antibody (anti-ssDNA), anti-double stranded DNA antibody (anti-dsDNA), anti-Sm antibody, anti-cardiolipin antibody, etc. appeaer in blood of SLE patients. Also rheumatoid factors are found to be noted in those with rheumatoid arthritis (RA); anti-SS-A antibody, anti-SS-B antibody and anti-mitochondrial antibody in those with Sjogren's syndrome (SjS); anti-scl antibody and anti-single stranded DNA antibody in progressive systemic sclerosis (PSS); and anti-RNP antibody in those with mixed connective tissue diseases (MCTD). At this point of time, there are many unclear points regarding relationship between the induction of these antibodies and significance in occurrence of the diseases. However, it is an important means for diagnosis of the diseases and knowledge of change in prognosis to determine the antibodies.
In order to detect these various autoantibodies, immunodiffusion test, counter immunoelectrophoresis, hemagglutination test, radioimmunoassay (RIA), enzyme immunoassay (EIA) and latex agglutination test, etc. have been developed depending on antigenic specificities of antibodies, and biochemical properties and physicochemical properties of antigens.
Turning to a method for determination of the aforesaid anti-cardiolipin antibodies, immunoprecipitation was firstly developed as diagnosis (mass screening) of syphilis in the late 1940's. In the diagnosis using this method, however, there was a problem that sera from patients with systemic lupus erythematosus (SLE) or some autoimmune diseases were shown to be biological false-positive (BFP), in addition to poor sensitivity. In recent years, a high concentration of anti-cardiolipin antibodies was found to be induced in blood of young female patients with lupus-like syndromes showing the symptom of thrombosis in the artery and vein, miscarriage or thrombocytopenia. Great attention has thus been given to clinical significance in diagnosis of these diseases by determining anti-cardiolipin antibodies.
Under such a situation, great expectations have been entertained of highly sensitive and accurate methods, instead of conventional assay such as non-specific determination of lupus anticoagulant or determination of anti-cardiolipin antibodies by immunoprecipitation and hemagglutination in which biological false-positive is noted with high frequence [Proctor, R. R. & Rapaport, S. L., Am. J. Clin. Pathol., 36, 212 (1961); Exner, T. et al., British Haematol., 40, 143 (1978); Margolios, A., Medicine, 40, 145 (1961)]. In 1983, RIA for determination of anti-cardiolipin antibodies was developed by Harris et al. [Harris, E. N., et al., Lancet, 1211-1214 (1983)]. Then, development of EIA was followed by non-competitive method (i.e., a sandwich method) using an antibody labeled with an enzyme, such as alkaline phosphatase. Determination of anti-cardiolipin antibodies by the EIA involved in the system is also quantitative and simple and, seems to be suitable for diagnosis and course observation of collagen diseases such as SLE, etc. In addition, this method is also used for analysis on specificity of anti-cardiolipin antibodies in the field of basic medical science. A strong view has been recently taken that among the region of collagen diseases, especially recurrent abortion would be caused by thrombosis due to induction of antiphospholipid antibodies such as anti-cardiolipin antibodies, etc. (antiphospholipid syndrome). It has thus been expected also in the field of obstetrics and gynecology to determine anti-cardiolipin antibodies.
Various subclasses (i.e., IgG, IgM, IgA, etc.) of anti-cardiolipin antibodies are observed in patients with collagen-diseases and infectious diseases, but IgG or IgM appears frequently in SLE patients.
Further in dsDNA, ssDNA, cardiolipin, phosphatidylserine, phosphatidylinositol, etc. which are seemed to be specific antigens to the respective antibodies specifically raised in sera of patients with antiphospholipid syndrome, similar molecular structures are present at the restricted polar site localizing around phosphodiesters thereof. Discussions have been currently made as to if these antibodies are identical or not.
As stated above, RIA and EIA for determining anti-cardiolipin antibodies have been developed as the assay system for diagnosis of antiphospholipid syndromes including SLE, recurrent abortion, etc. However, these assay methods still encounter the following problems.
That is, where sera of patients with SLE, etc. are provided for the assay, the method using an antigen-bound solid phase carrier sometimes involves difficulty in quantitatively determining antibodies specific to cardiolipin due to the nonspecific adsorption except for using appropriate blocking agent. Where fetal bovine serum (FBS) is conventionally used as a blocking agent, it is impossible to quantitatively determine differentially anti-cardiolipin antibodies derived from SLE and anti-cardiolipin antibodies caused by exogenous origin (i.e., infectious diseases, etc.). Any blocking agents suited for such a purpose have not been found yet.